Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peritoneal cells were derived from a patient (PK) with adenocarcinoma of the colon during the course of cisplatin/5-fluorouracil (5-FUra) treatment. Resistance to cisplatin and 5-FUra, characterized by a lack of response to chemotherapy and continued growth of the tumor, was concomitantly associated with a 2-4-fold increase in DNA copy number for dTMP synthase and dihydrofolate reductase. There was a corresponding amplification in DNA copy number of the c-myc (2X), H-ras (4X), and c-fos (15X) oncogenes. Cytogenetic studies revealed an iso (13q) chromosome, but failed to show any double minutes or homogeneously staining regions. In addition, drug-resistant tumor cells from PK and another patient (HG) displayed enhanced expression of dTMP synthase, c-fos and DNA polymerase beta when compared to normal colon tissue and the HCT8 human colon carcinoma cell line. These results suggest that elevated oncogene DNA and gene expression may be involved in the development of cisplatin resistance.
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PMID:Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil. 214 97

Biochemical differences were demonstrated between two cell lines derived from a human colon carcinoma (HCT8), one sensitive (HCT8S), and one 4.3-fold resistant to cisplatin (HCT8DDP). The cisplatin-resistant cell line overexpressed five enzymes (dihydrofolate reductase, thymidine 5'-monophosphate synthase, thymidine kinase, and DNA polymerase alpha and beta) believed to be important for DNA replicative and repair synthesis. In addition, the c-fos and c-H-ras oncogenes were also overexpressed in the HCT8DDP cells. This apparent overexpression was not associated with increases in gene copy number, it was related, however, to increased mRNA content. Expression of these key enzymes may be a significant factor in the development of clinical resistance to cisplatin. Further, these specific changes in cellular metabolism associated with cisplatin resistance may be exploited by the use of nucleoside analogues.
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PMID:Overexpression of DNA replication and repair enzymes in cisplatin-resistant human colon carcinoma HCT8 cells and circumvention by azidothymidine. 253 92

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.
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PMID:Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction. 269 61

Prior reports demonstrated more than additive cytotoxic effects of cis-diamminedichloroplatinum(II) (CDDP) and 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells. We have extended these findings by analyzing mechanisms that may underlie the effect of ara-C on CDDP-induced cytotoxicity. In contrast to a previous study, ara-C neither enhances DNA interstrand cross-link formation by CDDP nor affects the excision of platinum from DNA. Features peculiar to ara-C, such as its misincorporation into DNA, probably contribute since more than additive cytotoxic effects do not occur by combinations of CDDP with inhibitors of DNA synthesis that are not incorporated into DNA. Also, while ara-C does not significantly enhance the degree of inhibition of DNA synthesis caused by CDDP, the recovery of DNA synthesis after drug removal is significantly slowed when cells are exposed to both drugs. These findings contrast with those obtained with CDDP and aphidicolin (the latter agent resembles ara-C in competing with dCTP for binding to DNA polymerase alpha but, unlike ara-C, is not incorporated into DNA). Lastly, ara-C is incorporated into LoVo cell DNA undergoing replicative synthesis as well as into DNA undergoing repair synthesis after CDDP-induced induced DNA damage.
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PMID:Interactions of cis-diamminedichloroplatinum(II) with 1-beta-D-arabinofuranosylcytosine in LoVo colon carcinoma cells. 310 9

We categorized established human colon carcinoma cell lines into three biological groups. Our studies were performed on six colorectal cancer lines representing the proposed three groups. Group I consisted of two lines designated LoVo and SW 48; Group II comprised two lines called SW 480 and SW 620; and Group III was represented by lines SW 403 and SW 1116. Group I consisted of the most differentiated cells. This differentiation encompassed morphological markers, gland and signet ring formation, and ciliary development. The outstanding morphological characteristic of Group III was the development of numerous multinucleated giant cells. The range and modal chromosome number increased from Group I to Group III, a change reflected by the higher DNA content of these cells as measured by flow cytometry. Carcinoembryonic antigen synthesis was maximal for Group III and virtually absent for Group II. The number of clonogenic cells decreased from Group I to Group III, while the proportion of nonproliferating cells calculated both by experiments using continuous labeling with tritiated thymidine, and by the primer-available alpha-DNA polymerase index, increased from Group I through Group III. Another important cytokinetic difference was that Group I had an exponential cell cycle stage distribution not seen for the other groups. Cells in Group I were easily propagated in athymic (nude) rats by s.c. injection; cells in Group II injected s.c. grew for about 30 days and then regressed spontaneously. Cells in Group III could only be grown when inoculated intracerebrally. Thus, our studies have now confirmed and extended the hypothesis that cultured human colorectal carcinomas can be separated into at least three groups on the basis of morphological differentiation, chromatin distribution, carcinoembryonic antigen production, cytokinetic properties, and xenograft propagation. Perhaps this classification is just the tip of the iceberg, and future studies will determine the existence of additional groups or subgroups on the basis of other markers. However, at present it appears established that malignant cells with a common histological origin in the gut express their phenotypic potential in a sufficiently discrete manner as to permit their classification into distinct biological groups. Thus, the stage is set for extrapolating this in vitro classification for an in vivo segregation of human colorectal tumors into categories with specific properties and diverse prognosis.
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PMID:Cellular discriminants for a biological classification of human colon carcinoma. 646 84

Expression of the TS and DNA polymerase beta genes as a drug resistant-gene were compared with the human colon carcinoma cell line HCT8S and a subline that was 4.5 fold resistant to cisplatin. Resistant cells (HCT8DDP) exhibited 3.4 fold and 2.5 fold increase in mRNA for both TS and DNA polymerase beta gene when compared with the parent cells by Northern blotting analysis, respectively. The RT (reverse transcription)-PCR (polymerase chain reaction) method has been modified to quantify a sequence of a drug resistance gene. This method exhibited greater sensitivity than conventional methods (Northern blotting analysis), requiring less than 1 fetogram of mRNA from cell lines. The RT-PCR assay could be an effective device in the early detection of resistance to chemotherapy.
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PMID:[Detection of drug resistant-gene in cisplatin-resistant colon carcinoma cells by RT-PCR assay. I]. 835 15

DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific of the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase beta, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA replication by intercalative (e.g., actinomycin D) and nonintercalative (e.g., hydroxyurea) mechanisms. The transcriptional responses were measured as a function of the accumulation of CAT protein using antibodies against CAT protein in a standard ELISA. Endogenous cellular responses were evaluated for a number of the genes represented in the assay at both the mRNA and protein levels by Northern and Western blot analysis, respectively. These data corroborate the stress-induced responses measured by CAT ELISA in the CAT-Tox (D) assay, demonstrating the usefulness of this assay as a rapid and sensitive method for detection of DNA damaging agents in human cells.
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PMID:Stress responses to DNA damaging agents in the human colon carcinoma cell line, RKO. 895 Mar 45

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.
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PMID:Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay. 933 81

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange. In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins.
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PMID:Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers. 1076 Aug 17

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
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PMID:Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff. 1080 40


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